Department of Chemistry , University of California , Riverside , California 92521-0403 , United States.
ACS Chem Biol. 2019 Aug 16;14(8):1708-1716. doi: 10.1021/acschembio.9b00129. Epub 2019 Aug 1.
Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in -alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four -alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, -propyl, or butyl group) on DNA replication in human cells. We find that replication through the -alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ζ or Pol η. Genetic ablation of Pol ι, Pol κ, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G → A transition and G → T transversion mutations for these lesions. Moreover, further depletion of Pol ζ in Pol κ- or Pol ι-deficient cells gives rise to elevated rates of G → A and G → T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the -alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.
内源性代谢物和外源性化学物质可以诱导 DNA 上的共价修饰，产生 DNA 损伤。鸟嘌呤的修饰被证明是 DNA 中常见的烷基化位点；然而，对于烷基在 -烷化 dG 损伤中大小对细胞 DNA 复制的影响，以及跨损伤合成（TLS）聚合酶如何调节这些损伤在人类细胞中的 DNA 复制，人们知之甚少。为了回答这些问题，我们采用一种稳健的穿梭载体方法来研究四种 -烷化 dG 损伤（即烷基为甲基、乙基、-丙基或丁基）对人类细胞中 DNA 复制的影响。我们发现，在 HEK293T 细胞或同源 CRISPR 工程细胞中，通过 -烷化 dG 损伤的复制非常高效和准确，这些细胞缺乏聚合酶（Pol）ζ或 Pol η。然而，Pol ι、Pol κ 或 Rev1 的基因缺失会导致绕过效率降低，并引发这些损伤的大量 G→A 转换和 G→T 颠换突变。此外，在 Pol κ 或 Pol ι 缺陷细胞中进一步耗尽 Pol ζ 会导致 G→A 和 G→T 突变率升高和绕过效率显著降低。总之，我们证明了特定的 TLS 聚合酶有助于无差错地复制 -烷化 dG 损伤，并且我们发现这些损伤中的长烷基链会降低 DNA 复制的绕过效率和保真度。
